2,3,7,8-Tetrachlorodibenzo-p-dioxin: a potent inducer of -aminolevulinic acid synthetase.

2,3,7,8-Tetrachlorodibenzo-p-dioxin, a toxic contaminant frequently formed during the synthesis of the herbicide 2,4,5-trichlorophenoxyacetic acid, was shown to be a potent inducer of hepatic delta-aminolevulinic acid synthetase in the chick embryo. As little as 4.66 x 10(-12) mole of the contaminant per egg produces a significant increase in the activity of the enzyme. Induction of the enzyme is related to the dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin and, in contrast to that produced with other drugs, is prolonged in time, with 70 percent of the maximum induced activity present 5 days after a single dose. This contaminant is implicated as the likely causative agent in an outbreak of porphyria cutanea tarda in workers in a factory where 2,4,5-trichlorophenoxyacetic acid was being synthesized.

3,4,3',4'-Tetrachloro azoxybenzene and azobenzene: potent inducers of aryl hydrocarbon hydroxylase.

Two unwanted contaminants, 3,4,3',4'-tetrachloroazoxybenzene (TCAOB) and 3,4,3',4'-tetrachloroazobenzene (TCAB), formed in the commercial synthesis of 3,4-dichloroaniline or of herbicides made from 3,4-dichloroaniline, were responsible for three outbreaks of acne among chemical workers. TCAOB and TCAB are approximately isosteric to 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran, two well-known contaminants that cause acne. All four of these agents are potent inducers of hepatic aryl hydrocarbon hydroxylase activity and compete for stereospecific binding sites in the hepatic cytosol, which are thought to be the receptor sites for the induction of this enzyme. Among the chlorinated azoxy and azobenzenes, the potency of a congener to induce aryl hydrocarbon hydroxylase activity correlates with its binding affinity for the hepatic cytosol specific binding sites and its capacity to induce acne; this relation between structure and activity parallels that observed for the chlorinated dibenzo-p-dioxins and dibenzofurans.

Hormone synthesis and secretion by rat parathyroid glands in tissue culture.

Rat parathyroid glands maintained in organ culture secrete biologically active parathyroid hormone (PTH) and synthesize and secrete labeled proteins from (3)H- or (14)C-labeled amino acids added to the medium. The amounts of biological activity and labeled protein in the medium are both inversely proportional to the calcium concentration. Some of the labeled low molecular weight protein was identified as PTH which had been synthesized and secreted in culture by preliminary isolation on Sephadex G-100 columns and further purification using an antibody to bovine PTH which cross-reacted with rat PTH. The cross-reacting antibody inhibited the biological effects of rat PTH and caused hypocalcemia in intact rats. The antibody bound some of the labeled low molecular weight protein of the medium at neutral pH so that it migrated as a large molecular weight complex on Sephadex. Biologically active, labeled PTH was recovered by dissociation of this complex in acid and rechromatography.

Physiological and genetic analyses of inbred mouse strains with a type I iodothyronine 5' deiodinase deficiency.

Inbred mouse strains differ in their capacity to deiodinate iododioxin and iodothyronines, with strains segregating into high or low activity groups. Metabolism of iododioxin occurs via the type I iodothyronine 5'deiodinase (5'DI), one of two enzymes that metabolize thyroxine (T4) to 3,5,3'-triiodothyronine (T3). Recombinant inbred strains derived from crosses between high and low activity strains exhibit segregation characteristic of a single allele difference. Hepatic and renal 5'DI mRNA in a high (C57BL/6J) and low (C3H/HeJ) strain paralleled enzyme activity and concentration, in agreement with a recent report. 5'DI-deficient mice had twofold higher serum free T4 but normal free T3 and thyrotropin. Brown adipose tissue 5'DII was invariant between the two strains. Southern analyses using a 5'DI probe identified a restriction fragment length variant that segregated with 5'DI activity in 33 of 35 recombinant inbred strains derived from four different pairs of high and low activity parental strains. Recombination frequencies using previously mapped loci allowed assignment of the 5'DI gene to mouse chromosome 4 and identified its approximate chromosomal position. We propose the symbol Dio1 to denote the mouse 5'DI gene. Conserved linkage between this segment of mouse chromosome 4 and human HSA1p predicts this location for human Dio1.

An estimate of the maximum in vivo covalent binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to rat liver protein, ribosomal RNA, and DNA.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an extraordinarily potent toxin, has recently been found to be a potent carcinogen producing mucosal, lung, and liver tumors in female rats. In light of this carcinogenicity, we reexamined the in vivo covalent binding of [3H]TCDD to rat liver macromolecules. Immature Sprague-Dawley rats, receiving [3H]TCDD (0.87 mCi/kg; specific activity, 39 Ci/mmol) concentrated 18 to 64% of the total administered dose in their livers, but virtually all of this radioactivity (greater than 99.9%) was extractable. The maximum unextractable radioactivity was: protein, 60 pmol TCDD per mol of amino acid residue; rRNA, 12 pmol TCDD per mol of nucleotide residue; and DNA, 6 pmol TCDD per mol of nucleotide residue. If one assumes that this small residual amount of radioactivity represents covalent binding, this binding is 4 to 6 orders of magnitude lower than that of most chemical carcinogens, and the binding to DNA is equivalent to one molecule of TCDD per DNA, equivalent to 35 cells. The results suggest it is unlikely that TCDD-induced oncogenesis is through a mechanism of covalent binding to DNA and somatic mutation.

Quantitative evaluation of the promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxin of hepatocarcinogenesis from diethylnitrosamine.

In order to test the potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as a promoter of hepatocarcinogenesis, rats which had received a single 10-mg/kg dose of diethylnitrosamine (DEN) following partial hepatectomy were given TCDD (0.14 and 1.4 micrograms/kg s.c. once every 2 weeks) for 7 months. Animals which received (a) only a single initiating dose of DEN after partial hepatectomy and no further treatment of (b) TCDD alone with no initiating dose of DEN exhibited relatively few enzyme-altered foci and no hepatocellular carcinomas. However, animals initiated with DEN and then given TCDD had a marked increase in enzyme-altered foci. At the higher dose of TCDD, hepatocellular carcinomas were present in five of seven rats. By means of three different enzyme markers used to evaluate the phenotypes of the enzyme-altered foci, a distinct phenotype heterogeneity of the foci was noted with a shift towards phenotypes exhibiting a greater deviation from normal liver when TCDD was given following DEN-partial hepatectomy. Quantitation of the numbers of enzyme-altered foci was performed by relating measurements made from two-dimensional tissue sections to the numbers of foci per unit volume of liver using relationships established in the field of stereology. The total volume of the liver occupied by the enzyme-altered foci, but not their number, increased with the dose of TCDD administered following DEN-partial hepatectomy. These studies demonstrate that TCDD is a potent promoting agent for hepatocarcinogenesis.

Strong evidence from studies with brachymorphic mice and pentachlorophenol that 1'-sulfoöxysafrole is the major ultimate electrophilic and carcinogenic metabolite of 1'-hydroxysafrole in mouse liver.

The role of sulfation of 1'-hydroxysafrole in the formation of hepatic macromolecular adducts and in hepatic tumor formation in mice given 1'-hydroxysafrole was investigated by the use of: (a) mice treated with the specific sulfotransferase inhibitor pentachlorophenol; and (b) brachymorphic mice, which are characterized by a deficiency in the hepatic synthesis of 3'-phosphoadenosine 5'-phosphosulfate. Cytosolic sulfotransferase activity for 1'-hydroxysafrole in both mouse and rat liver was significantly inhibited by 10 microM pentachlorophenol, usually by greater than 90%. Prior administration of nontoxic amounts of pentachlorophenol, either in the diet of adult female CD-1 mice or by i.p. injection of 12-day-old male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice, resulted in an 85% decrease in the level of adducts formed from 1'-hydroxysafrole in hepatic DNA and RNA as compared to those of non-pentachlorophenol-treated animals. Likewise, the chronic administration of a nontoxic level of pentachlorophenol in the diet of adult female CD-1 mice strongly inhibited hepatic tumor induction by long-term dietary administration of either safrole or 1'-hydroxysafrole. Initiation of hepatic tumors by a single i.p. injection of 1'-hydroxysafrole to 12-day-old male B6C3F1 mice was strongly inhibited by prior treatment with pentachlorophenol. Under these conditions, the hepatocarcinogenicity of diethylnitrosamine was not inhibited by pentachlorophenol. Supplementation with adenosine triphosphate and sulfate of hepatic cytosols from adult female or 12-day-old brachymorphic progeny of a B6C3 background outbred to B6C3F1 mice (B6C3F2), of either sex, resulted in 5- to 10-fold less binding of 1'-hydroxysafrole to added RNA than when cytosols from phenotypically normal B6C3F2 mice were used. On administration of [3H]-1'-hydroxysafrole to adult female or 12-day-old brachymorphic B6C3F2 mice of either sex, the levels of hepatic DNA and RNA adducts were 7- to 12-fold lower than those obtained in phenotypically normal B6C3F2 mice of the same age and sex. Brachymorphic mice were also much less responsive than their phenotypically normal littermates to the induction of liver tumors by 1'-hydroxysafrole; lower incidences were observed both when the carcinogen was fed chronically to adult females and when it was administered to males only prior to weaning. Thus, all of these data strongly support the conclusion that 1'-sulfoöxysafrole is the major ultimate electrophilic and tumor-initiating metabolite of 1'-hydroxysafrole.

Histologic changes produced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the skin of mice carrying mutations that affect the integument.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) produces epidermal hyperplasia and hyperkeratosis, squamous metaplasia of the sebaceous gland, and keratinized cyst formation in 8 strains of mice with the recessive mutation, hairless (hr/hr). The extent of these histologic changes is dependent on the genetic background. No cutaneous lesions are produced in haired (hr/+) mice. In examination of mice with 7 other mutations affecting the integument, TCDD produced similar histologic skin changes in cryptothrix, nude, plucked, and atrichosis; a marginal squamous metaplasia of sebaceous glands in Repeated epilation, and had no effect in fur deficient and Naked mutants. These genetically determined epidermal responses are discussed in light of the mechanism of action of TCDD.

Impairment of the selenoenzyme type I iodothyronine deiodinase in C3H/He mice.

Type I iodothyronine deiodinase (ID-I) activity is impaired in C3H/He (C3H) mice compared with BALB/c and C57BL/6N (C57) mice. In this study we compared ID-I activity and protein labeling with N-bromoacetyl(-)[125I]T3 (BrAc[125I]T3) or 75Se in liver microsomes of C3H and C57 mice. Hepatic ID-I activity in C3H mice was highly variable with a median of only 18% of that in C57 mice. However, C3H mice had normal serum T4 and T3 levels, although serum reverse T3 was increased. The 28-kilodalton (kDa) ID-I protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of BrAc[125I]T3-labeled microsomes. Labeling of this protein was virtually undetectable in C3H samples with low enzyme activity. ID-I activity in liver microsomes was strongly decreased in Se-deficient mice, which was paralleled by a drastic decrease in BrAc[125I]T3-labeling of the 28-kDa band compared with control mice. Labeling of ID-I with 75Se was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of liver microsomes of [75Se]selenite-injected mice. 75Se labeling of the 28-kDa band was markedly higher in Se-deficient than in control mice and was also markedly higher in C57 than in C3H mice. Finally, liver ID-I messenger RNA (mRNA) was measured on Northern blots using a rat ID-I complementary DNA probe. Messenger RNA levels correlated strongly with ID-I activity, showing a significant decrease in C3H mice. We conclude that in mice, like in rats and humans, ID-I is a selenoprotein. ID-I activity is impaired in C3H mice because of decreased transcription of the ID-I gene or reduced stability of the mRNA.

Effect of the brachymorphic trait in mice on xenobiotic sulfate ester formation.

Mice carrying the recessive mutation brachymorphic have been shown previously to have a reduced capacity to synthesize 3'-phosphoadenosine-5'-phosphosulfate (PAPS), the required coenzyme in sulfation reactions [K. Sugahara and N. Schwartz, Proc. natn. Acad. Sci. U.S.A. 76, 6615 (1979)]. The capacity of the liver cytosol fractions from brachymorphic (bm/bm) mice or their phenotypically normal littermates (+/+ or +/bm) to catalyze the formation of sulfate esters of [3H]estrone and [14C]p-nitrophenol in vitro was determined. When PAPS was added to the reaction, the rates of sulfate ester formation catalyzed by the two cytosol fractions were similar. In contrast, when PAPS was generated in situ from ATP and SO(4)2-, the rates of sulfate ester formation catalyzed by the brachymorphic cytosol were only 4-22% of the rates catalyzed by the cytosol fraction from normal mice. The hepatic cytosol fraction from brachymorphic mice incorporated less 35SO(4)2- into PAPS than that catalyzed by cytosol of normal mice. [14C]p-Nitrophenol (1.5 mumoles/kg) was eliminated from brachymorphic and normal mice as urinary conjugates; in normal mice, 73% of the urinary radioactivity was p-nitrophenyl sulfate, while in the brachymorphic mice only 33% of the urinary excretion was the sulfate ester. Brachymorphic mice have a reduced capacity for synthesizing sulfate esters of xenobiotics in vitro and in vivo, which is attributable to their reduced synthesis of PAPS.

The trouble with TEFs.

Comments on Van den Berg, et al. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife. Environ Health Perspect 106:775-792 (1998)

Chloracne from manufacture of a new herbicide.

Forty-one chemical company workers had chloracne as a result of exposure to 3,4,3',4'-tetrachloroazoxybenzene (TCAB), an extraneous intermediate produced during the manufacture of a new herbicide. There was no laboratory evidence of significant hepatotoxicity or porphyria during the short time the herbicide was produced. An acnegenic when applied to the rabbit ear, TCAB is also a potent inducer of the microsomal enzyme aryl hydrocarbon hydroxylase. Prevention of chlorance is a difficult industrial engineering task and treatment of the disease is even more perplexing.

An isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus.

An isolated epizootic of a highly fatal feline calicivirus (FCV) infection, manifested in its severest form by a systemic hemorrhagic-like fever, occurred over a 1-month period among six cats owned by two different employees and a client of a private veterinary practice. The infection may have started with an unowned shelter kitten that was hospitalized during this same period for a severe atypical upper respiratory infection. The causative agent was isolated from blood and nasal swabs from two cats; the electron microscopic appearance was typical for FCV and capsid gene sequencing showed it to be genetically similar to other less pathogenic field strains. An identical disease syndrome was recreated in laboratory cats through oral inoculation with tissue culture grown virus. During the course of transmission studies in experimental cats, the agent was inadvertently spread by caretakers to an adjoining room containing a group of four normal adult cats. One of the four older cats was found dead and a second was moribund within 48-72h in spite of symptomatic treatment; lesions in these animals were similar to those of the field cats but with the added feature of severe pancreatitis. The mortality in field cats, deliberately infected laboratory cats, and inadvertently infected laboratory cats ranged from 33-50%. This new isolate of calicivirus, named FCV-Ari, was neutralized at negligible to low titer by antiserum against the universal FCV-F9 vaccine strain. Cats orally immunized with FCV-F9, and then challenge-exposed shortly thereafter with FCV-Ari, developed a milder self-limiting form of disease, indicating partial protection. However, all of the field cats, including the three that died, had been previously immunized with parenteral FCV-F9 vaccine. FCV-Ari caused a disease that was reminiscent of Rabbit Hemorrhagic Disease, a highly fatal calicivirus infection of older rabbits.

Photoaffinity labelling of the Ah receptor.

A series of halodibenzo-p-dioxins bearing the arylazide photolabile functional group were synthesized and tested as photoaffinity labels for the Ah receptor. 2-Azido-3-iodo-7,8-dibromodibenzo-p-dioxin (KD = 0.76 X 10(-9) M) was selected for radiosynthesis. Analysis of the 125I-photoaffinity-labelled proteins in mouse-liver cytosol by denaturing gel electrophoresis revealed two peptides which had apparent molecular masses of 95,000 and 70,000 daltons respectively, were labelled in an approximately 1:1 ratio and were selectively labelled at low concentrations of the photoaffinity ligand (0.05 KD = 0.04 X 10(-9) M). In addition, their labelling was inhibited by co-incubation with an excess of unlabelled ligand. On chromatographic separation under non-denaturing conditions, these two peptides co-migrated. These studies suggest that the Ah receptor in mouse liver cytosol is a heterodimer composed of two non-covalently bound peptides (95 K and 70 K) which each have a ligand binding site.

Altering the thermal resistance of foodborne bacterial pathogens with an eggshell membrane waste by-product.

Eggshells from egg-breaking operations are a significant waste disposal problem. Thus, the development of value-added by-products from this waste would be welcomed by the industry. The ability of extracted eggshell membranes containing, several bacteriolytic enzymes (i.e., lysozyme and beta-N-acetylglucosaminidase) or other membrane components to alter the thermal resistance of gram-positive and gram-negative bacterial pathogens was evaluated. Mid-log phase cells of Salmonella Enteritidis (SE), Salmonella Typhimurium (ST), Escherichia coli O157:H7 (EC), Listeria monocytogenes Scott A (LM), and Staphylococcus aureus (SA) were suspended in 100 ml of 0.1% peptone water (pH 6.9, 10(7-8) CFU/ml) containing either 0 (control) or 10 g of an eggshell membrane extract and incubated at 37 degrees C for 45 min. Following exposure, membrane-free samples (1.5 ml) were heated in a 56 degrees C (LM, SA), 54 degrees C (SE, ST), or 52 degrees C (EC) water bath from 0 to 14 min in sealed glass reaction vials (12 by 32 mm), and the survivors were recovered on brain heart infusion agar. Population reductions ranging from 27.6% (SA) to 99.8% (LM) (ST, 43.8%; SE, 47.5%; EC, 71.8%) were observed for cells treated for 45 min with extracted membrane, as compared to controls. D-value reductions ranging from 0 (LM) to 87.2% (SE) (SA, 36.7%; EC, 83.3%; ST, 86.3%) were observed when membrane-treated cells were subsequently heat inactivated. The effects of exposure pH, time, temperature, and organic load on membrane activity were also evaluated with Salmonella Typhimurium. Exposure pH (5.0 versus 6.9), time (15 versus 45 min), and temperature (4 degrees C versus 37 degrees C) did not significantly reduce the impact of eggshell membranes on D-values. However, the presence of organic matter (0.1% peptone water versus skim milk) significantly reduced the thermal resistance-reducing capacity of the membranes. These preliminary findings provide information on the potential use of extracted eggshell membranes to alter bacterial heat resistance.

Diagnostic features of clinical neurologic feline infectious peritonitis.

Feline infectious peritonitis (FIP) is a fatal Arthus-type immune response of cats to infection with FIP virus, a mutant of the ubiquitous feline enteric coronavirus (FECV). The disease may occur systemically or in any single organ system, and primary neurologic disease is a common subset of such manifestations. We examined 16 domestic cats with clinical neurologic FIP and 8 control cats with nonneurologic FIP, with the intention of identifying the ante- and postmortem diagnostic tests that most contribute to accurate diagnosis. Of the 16 cats with neurologic FIP, 15 were less than 2 years of age and all 16 originated from large multiple-cat households. The most useful antemortem indicators of disease were positive anti-coronavirus IgG titer in cerebrospinal fluid, high serum total protein concentration, and findings on magnetic resonance imaging suggesting periventricular contrast enhancement, ventricular dilatation, and hydrocephalus. Postmortem diagnosis was facilitated by FIP monoclonal antibody staining of affected tissue and coronavirus-specific polymerase chain reaction. Most cats with neurologic and ocular forms of FIP had patchy, focal lesions, suggesting that recently developed technologies described in this report may be useful for evaluation of cats with suspected FIP.

Disease outcome and cytokine responses in cats immunized with an avirulent feline infectious peritonitis virus (FIPV)-UCD1 and challenge-exposed with virulent FIPV-UCD8.

Eight cats were immunized with an avirulent strain of feline infectious peritonitis virus (FIPV)-UCD1, then challenge-exposed to a highly virulent cat passaged strain (FIPV-UCD8). Th1 and Th2 cytokine profiles in the peripheral blood mononuclear cells (PBMCs) were measured throughout in the experiment. No clinical signs of FIP were evident in the experimental cats after immunization. After challenge, the immunized cats demonstrated one of four clinical outcomes: (1) classical effusive FIP; (2) accelerated FIP; (3) non-effusive FIP, or (4) resistance to challenge. Only minor cytokine changes were observed following immunization, however, several cytokine changes occurred following challenge-exposure. The most noteworthy changes were in tumor necrosis factor-alpha (TNF-alpha) and interferon gamma (IFN-gamma) levels. Our preliminary findings suggest that immunity against FIP is associated with TNF-alpha and IFN-gamma response imbalance, with high TNF-alpha/low IFN-gamma mRNA responses favouring disease and low TNF-alpha/high IFN-gamma mRNA responses being indicative of immunity.

Common virus infections in cats, before and after being placed in shelters, with emphasis on feline enteric coronavirus.

The purpose of this study was to determine the origin and subsequent spread of feline calicivirus (FCV), feline herpesvirus (FHV), and feline enteric coronavirus (FECV) in cats relinquished to shelters. FCV was isolated from the oral fauces of 11% of healthy cats upon entry, and isolation rates were highest for kittens (33%). FHV shedding was very low (4%) at the time of entry and occurred mainly in juveniles. FECV shedding was also common among newly relinquished cats (33%), especially older kittens and juveniles (90%). The subsequent spread of all three viruses was rapid and efficient in the shelter environment. Fifteen percent of cats were shedding FCV, 52% FHV, and 60% FECV after 1 week. More detailed studies were done with FECV shedding, which could be accurately quantitated. The amounts of FECV shed by infected cats ranged from 10(2)to 10(16)particles/swab of feces. FECV shedding was several logs higher in young kittens with primary infection than adult cats with primary infections. The mean levels of FECV shedding among adults were the same for primary and chronic infections. Although shelters were not the primary source of these viruses for many relinquished cats, factors intrinsic to the shelter environment were critical in amplifying shedding and spread to susceptible individuals. Extrinsic factors were especially important for the spread of FHV and FECV. FHV shedding rates increased from 4% to 50% in 1 week's time. The speed and magnitude of the increase in FHV shedding suggested that there was reactivation of latent infections as well as acquisition of new infections. FECV shedding increased 10 to 1,000,000 fold in 1 week among cats that were already infected at entry, and more than one-half of initially negative cats were shedding FECV a week later. Feline calicivirus infection was the least likely to spread in the shelter. The infection rate only increased from 11 to 15% in 1 week.

An epizootic of highly virulent feline calicivirus disease in a hospital setting in New England.

This article reports an outbreak of 24 cases of an unusually virulent feline calicivirus (FCV) infection in a small animal hospital. The circumstances and disease signs were very similar to those recently described in an outbreak of FCV hemorrhagic disease in Northern California (Vet. Microbiol. 73 (2000) 281). The virus entered the facility through shelter cats showing upper respiratory signs. Affected cats manifested high fever, anorexia, labored respirations, oral ulceration, facial and limb edema, icterus, and pancreatitis. The infection spread rapidly among the patients by contaminated animal caretakers and hospital equipment. One case of fomite transmission from an employee to a housecat was documented. Prior vaccination, even with multiple doses of FCV-F9-based live calicivirus vaccine, was not protective. Affected cats often required extensive supportive care for 7-10 days, and the overall mortality from death and euthanasia was 32%. The strain of FCV responsible for this outbreak was genetically and serologically distinct from the FCV strain responsible for a similar epizootic and the FCV-F9 strain contained in most vaccines. Outbreaks of this type are being reported with increasing frequency, and are often associated with the practice of treating sick shelter cats in private practices. Similar to the present epizootic, outbreaks of FCV hemorrhagic disease have been self-limiting, but require prompt application of strict quarantine, isolation, personnel sanitation, and disinfection procedures.

Molecular, clinical, and pathologic comparison of two distinct strains of Haemobartonella felis in domestic cats.

OBJECTIVE: To characterize 2 strains of Haemobartonella felis by use of molecular techniques. ANIMALS: 35 specific-pathogen-free cats, 6 months to 4 years old. PROCEDURE: Intraperitoneal or IV inoculation with blood containing H felis small form (Hfsm, 18 cats) or H felis large form (Hflg, 11 cats); 6 cats were uninfected controls. Hfsm was evaluated for capability to cross-protect against the more virulent Hflg. Morphology of both strains was compared by light microscopy of Wright-Giemsa-stained blood smears, and the 16S rRNA genes were sequenced. RESULTS: Infection with Hflg induced signs of depression, fever, and severe macrocytic normochromic anemia with nucleated erythrocytes. More than 95% of erythrocytes were parasitized. Inoculation with Hfsm and uninfected control blood induced mild or no clinical signs and no hematologic abnormalities. Anti-H felis IgG was first detected on postinoculation day (PID) 21, and increased to maximal titer of 400 by PID 28. Reactivated infection was observed in 8 of 29 cats (4 Hfsm and 4 Hflg), with 5% parasitized erythrocytes during the later attack. On PID 8, Hflg-inoculated cats had positive results of polymerase chain reaction analysis (PCR) that persisted until cats were treated with doxycycline or oxytetracycline; Hfsm-inoculated cats had positive PCR results that persisted for duration of observation (3 months). CONCLUSIONS: Genetically and morphologically distinct strains of H felis infect cats in the field. The level of genetic difference suggested that these strains may be different species or genera. CLINICAL RELEVANCE: PCR is a critical diagnostic aid to detect occult Haemobartonella spp infection, as well as response to treatment and clearance of the organism.